Identification and characterization of a cDNA encoding a gametocyte-specific protein of the avian coccidial parasite Eimeria necatrix

Dandan Liu; Feiyan Wang; Liqin Cao; Lele Wang; Shijie Su; Zhaofeng Hou; Jinjun Xu; Junjie Hu; Jianping Tao

doi:10.1016/j.molbiopara.2020.111318

Identification and characterization of a cDNA encoding a gametocyte-specific protein of the avian coccidial parasite Eimeria necatrix

Mol Biochem Parasitol. 2020 Nov:240:111318. doi: 10.1016/j.molbiopara.2020.111318. Epub 2020 Sep 18.

Authors

Dandan Liu¹, Feiyan Wang², Liqin Cao³, Lele Wang⁴, Shijie Su⁵, Zhaofeng Hou⁶, Jinjun Xu⁷, Junjie Hu⁸, Jianping Tao⁹

Affiliations

¹ College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China; Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, China. Electronic address: ddliu@yzu.edu.cn.
² College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China; Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, China. Electronic address: mz120170969@yzu.edu.cn.
³ College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China; Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, China. Electronic address: caoliqin_0523@126.com.
⁴ College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China; Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, China. Electronic address: 1941055103@qq.com.
⁵ College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China; Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, China. Electronic address: ssjmxh@163.com.
⁶ College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China; Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, China. Electronic address: zfhou@yzu.edu.cn.
⁷ College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China; Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, China. Electronic address: jjxu@yzu.edu.cn.
⁸ Biology Department, Yunnan University, Kunming, 650500, China. Electronic address: jjhu@ynu.edu.cn.
⁹ College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China; Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, China. Electronic address: yzjptao@126.com.

PMID: 32956771
DOI: 10.1016/j.molbiopara.2020.111318

Abstract

Gametocyte proteins of Eimeria spp. are essential components of the oocyst wall, and some of these proteins have been analysed to identify targets of transmission-blocking vaccines against avian coccidiosis. In the present study, a cDNA from E. necatrix gametocytes was cloned and sequenced. The cDNA is 1473 bp in length and encodes a 490-amino-acid protein containing a tyrosine-serine (Tyr/Ser)-rich domain and a proline-methionine (Pro/Met)-rich domain. A quantitative real-time PCR (qPCR) analysis showed that the cDNA is expressed only during gametogenesis. A fragment containing the Tyr/Ser-rich domain (rEnGAM59) was expressed in Escherichia coli BL21 (DE3) cells. Immunoblotting showed that rEnGAM59 was recognized by the serum of convalescent chickens after infection with E. necatrix, and that an anti-rEnGAM59 antibody recognized a ∼59 kDa protein and two other proteins (∼35 kDa and ∼33 kDa) in gametocyte extracts. An immunofluorescence assay showed that the anti-rEnGAM59 antibody recognized wall-forming bodies in the macrogametocytes and oocyst walls. An in vivo vaccination and challenge trial was conducted to test the potential utility of rEnGAM59 as a vaccine. Immunized chickens performed better than the unimmunized and challenged (positive control) chickens. The intestinal lesion scores were significantly lower in the immunized groups than in the positive control group (P < 0.05). In contrast, the body weight gains (BWG) were significantly higher in the immunized groups than in the positive control group (P < 0.05). There were no significant differences in the lesion scores and BWG between the groups immunized with rEnGAM59 protein or with live oocysts (P> 0.05). Chickens immunized with rEnGAM59 protein had a significantly higher antigen-specific serum IgY response (P < 0.05). rEnGAM59 protein can be used as candidate antigen to develop a recombinant coccidiosis vaccine.

Keywords: Characterization; Clone; Eimeria necatrix; Expression; Gametocyte protein; cDNA.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Antibodies / immunology
Antigens, Protozoan / chemistry
Antigens, Protozoan / genetics
Antigens, Protozoan / immunology
Base Sequence
Cloning, Molecular*
Coccidiosis / immunology
Coccidiosis / parasitology
Coccidiosis / prevention & control
DNA, Complementary*
Eimeria / genetics*
Eimeria / immunology
Gene Expression*
Immunization
Protozoan Proteins / chemistry
Protozoan Proteins / genetics*
Protozoan Proteins / immunology
Protozoan Vaccines / immunology
Sequence Analysis, DNA

Substances

Antibodies
Antigens, Protozoan
DNA, Complementary
Protozoan Proteins
Protozoan Vaccines